The present invention is concerned with compositions capable of preventing, inhibiting or reducing restenosis (sometimes referred to in the art as xe2x80x9caccelerated arteriosclerosisxe2x80x9d and xe2x80x9cpost-angioplasty narrowingxe2x80x9d).
The following references are considered to be pertinent for the purpose of understanding the background of the present invention.
1. Waller, B. F., Orr, C. M, VanTassel J., et al. Clin-Cardiol. 20(2):153-60, (1997).
2. Anderson, W. D, King, S. Br., Curr-Opin-Cardiol., 11(6):583-90, (1996).
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4. Laurent S, Vanhoutte P, Cavero I, et al., Fundam. Clin. Pharmacol., 10(3):243-57, (1996).
5. Walsh, K., Perlman, Semin-Interv-Cardiol., 1(3):173-9, (1996).
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8. Hamon. M., Bauters, C., McFadden, E. P, et al., Eur. Heart J., 16:33s-48s, (1995).
9. Gottsauner-Wolf, M., Moliterno, D. J, Lincoff, A. M., Topol, E. J., Clin. Cardiol., 19:347-356, (1996).
10. Herrman, J. P. R., Herrnans, W. R. M., Vos, J., Serruys, P. W., Drugs, 46:18-52, (1993).
11. Leclerc, G., Voisine, P., Bouchard, M., Fleser, A., Martel, R. Elsevier Science, 722-724, (1995).
12. Topol, E., The NY Academy of Sciences, 225-277, (1997).
13. Fleisch, H., in: Bisphosphonates in bone disease. Parthenon Publishing Group Inc., pp. 184-186 (1997).
14. Mxc3x6nkkxc3x6nen, J., Taskinen, M., Auriola, S., Urtti, J. Drug Target, 2:299-308, (1994).
15. Kramsche, D. M., and Chan, C. T., Circ. Res., 42:562-572, (1978).
16. Braunwald, E. Heart Disease in: A textbook of cardiovascular medicine; 5th Ed., W. B. Saunders Company: Philadelphia, (1997).
17. Gennaro Alfonso, R. Remington, in: The Science and Practice of Pharmacy, Mack Publishing, Easton Pa., 19th ed., (1995).
18. Mxc3x6nkkxc3x6nen, J., and Heath, T. D., Calcif. Tissue Int., 53:139-145, (1993).
19. M. Donbrow in: Microencapsulation and Nanoparticles in Medicine and Pharmacy, CRC Press, Boca Raton, Fla., p. 347.
The above references will be acknowledged in the text below by indicating their number (as shown in brackets) from the above list.
Over the past decade, mechanical means of achieving revascularization of obstructive atherosclerotic vessels have been greatly improved. Percutaneous transluminal coronary angioplasty (PTCA) procedures include, but are not limited to, balloon dilatation, excisional atherectomy, endoluminal stenting, rotablation, and laser ablation. However, revascularization induces thrombosis and neointimal hyperplasia, which in turn cause restenosis in a substantial proportion of coronary arteries after successful balloon angioplasty and in aortacoronary saphenous vein bypass graft and other coronary grafts. Furthermore, intimal hyperplasia causes restenosis in many superficial femoral angioplasties, carotid endarterectomies, and femoro-distal vein bypasses. The introduction of endovascular stents has contributed to reduce the incidence of restenosis, but this problem still remains significant(1-9). Despite extensive research on the incidence, timing, mechanisms and pharmacological interventions in humans and animal models, to date no therapy exists which consistently prevents coronary restenosis(10-12). Compositions and methods for the reduction or prevention of restenosis are still today greatly desired.
Bisphosphonates (BP) (formerly called diphosphonates) are compounds characterized by two C-P bonds. If the two bonds are located on the same carbon atom (P-C-P) they are termed geminal bisphosphonates. The BPs are analogs of the endogenous inorganic pyrophosphate which is involved in the regulation of bone formation and resorption. The term bisphosphonates is generally used for geminal and non-geminal bisphosphonates. The BPs and pyrophosphates may at times form together polymeric chains. BPs act on bone because of their affinity for bone mineral and are potent inhibitors of bone resorption and ectopic calcification. BPs or pyrophosphate have been used clinically mainly as (a) antiosteolytic agents in patients with increased bone destruction, especially Paget""s disease, tumor bone disease and osteoporosis; (b) skeletal markers for diagnostic purposes (linked to 99mTc); (c) inhibitors of calcification in patients with ectopic calcification and ossification, and (d) antitartar agents added to toothpaste(13).
In accordance with the present invention, a BP or pyrophosphate (collectively herein: xe2x80x9cactive ingredientxe2x80x9d) is used for the treatment or prevention of vascular restenosis. The term bisphosphonate (BP) as used herein denotes both geminal and non-geminal bisphosphonates. The term xe2x80x9cactive ingredientxe2x80x9d encompasses in its scope also polymeric chains of the BPs or pyrophosphate, particularly such chains consisting of up to 40 BP monomers. Preferred active ingredients are compounds of the following formula (I): 
wherein R represents O or a CR1R2 group;
R1 is H, OH or a halogen group; and
R2 is halogen; linear or branched C1-C10 alkyl or C2-C10 alkenyl optionally substituted by heteroaryl or heterocyclyl C1-C10 alkylamino or C3-C8 cycloalkylamino where the amino may be a primary, secondary or tertiary; xe2x80x94NHY where Y is hydrogen, Cs-Cg cycloalkyi, aryl or heteroaryl; or R2 is xe2x80x94SZ where Z is chlorosubstituted phenyl or pyridinyl.
The present invention thus provides the use of said active ingredient, a complex of said active ingredient or a pharmaceutically acceptable salt or ester thereof, for the preparation of a medicament for the prevention or treatment of vascular restenosis.
The present invention also provides a method of treatment of restenosis, comprising administering to an individual in need thereof an effective amount of said active ingredient, a complex thereof or a pharmaceutically acceptable salt or ester thereof.
The present invention still further provides a pharmaceutical composition for the prevention or treatment of restenosis, comprising as an active ingredient, an effective amount of said active ingredient, a pharmaceutically acceptable free acid, a complex or a salt thereof, optionally together with a pharmaceutically acceptable carrier or diluent. A particularly preferred carrier is a liposome preparation.
The term xe2x80x9ceffective amountxe2x80x9d means to denote an amount of the active ingredient which is effective in achieving the desired therapeutic result, namely prevention or reduction of vascular restenosis. The effective amount may depend on a number of factors including: weight and gender of the treated individual; the type of medical procedure, e.g. whether the vascular restenosis to be inhibited is following balloon angioplasty, balloon angioplasty followed by deployment of a stent, etc.; the mode of administration of the active ingredient (namely whether it is administered systemically or directly to the site); the type of carrier being used (e.g. whether it is a carrier that rapidly releases the active ingredient or a carrier that releases it over a period of time); the therapeutic regime (e.g. whether the active ingredient is administered once daily, several times a day or once every few days); clinical factors influencing the rate of development of restenosis such as diabetes, smoking, hypercholesterolemia, renal diseases, etc.; anatomical factors such as whether there is severe preangioplasty stenosis, total occlusion, left anterior descending coronary artery location, saphenous vein graft lesion, long lesions, multivessel or multilesion PTCA; on the dosage form of the composition; etc. Moreover, procedural variables may also have bearing on the dosage such as greater residual stenosis following PTCA, severe dissection, intimal tear, appropriate size of balloon, and the presence of thrombus. The artisan, by routine type experimentation should have no substantial difficulties in determining the effective amount in each case.
The invention is applicable for the prevention, reduction or treatment of vascular restenosis and mainly, but not limited to, coronary restenois after angioplasty. Vascular restenosis results from various angioplasty procedures including balloon angioplasty, intravascular stent implantation or other methods of percutaneous angioplasty (including angioplasty of coronary arteries, carotid arteries, and other vessels amenable for angioplasty) as well as for restenosis resulting from vascular graft stenosis (e.g., following by-pass surgery)(16). In addition, the invention is also applicable for the prevention, reduction or treatment of vascular resteriosis in peripheral arteries and veins.
One exemplary application of the invention is to prevent and treat in-stent restenosis. It is a widely acceptable medical procedure to deploy a stent within a blood vessel within the framework an angioplastic procedure, to support the walls of the blood vessel. However, very often restenosis occurs notwithstanding the presence of the stent within the blood vessel. In accordance with the invention the above noted active ingredient may be administered, either systemically or directly to the site, in order to prevent or inhibit such restenosis. Potentially said active ingredient may be formulated in a manner allowing its incorporation onto the stent which will in fact yield administration of said active ingredient directly at the site. The active ingredient may be formulated in that manner, for example, by including it within a coating of the stent. Examples of coating are polymer coatings, e.g., made of polyurethane, or a gel.
The active ingredient used in accordance with the invention may be formulated into pharmaceutical compositions by any of the conventional techniques known in the art (see, for example, Alfonso el al., 1995(17)). The compositions may be prepared in various forms such as, capsules, tablets, aerosols, solutions, suspensions, or as a coating of a medical device such as a stent (see above), etc. In addition, the pharmaceutical compositions of the invention may be formulated in a form suitable for topical administration with a carrier or in a delivery form permitting penetration of the active ingredient through the skin and into the body in order to act systemically. The preferred administration form in each case will depend on the desired delivery mode, which is usually that which is the most physiologically compatible in accordance with the patient""s condition, other therapeutic treatments which the patient receives, etc. In accordance with a preferred embodiment of the invention, said active ingredient is formulated into a particulate form. This may be achieved by encapsulating or impregnating the active ingredient into particles, e.g., polymeric particles, lipid vesicles, or liposomes. Furthermore, such particles may be particles of polymerized active ingredient (see below). Particularly preferred are liposomal preparations of said active ingredient. The liposomes may be prepared by any of the methods known in the art (regarding liposome preparation methods see Mxc3x6nkkxc3x6nen et al., 1994(14) and Mxc3x6nkkxc3x6nen et al., (18). The liposomes may be positively charged, neutral or negatively charged (negatively charged liposomes being currently preferred), and may be single or multilamellar. At times, compositions comprising a combination of free active ingredient (i.e. non-encapsulated) and said active ingredient encapsulated within liposomes may also be used.
By a preferred embodiment of the invention, the active ingredient is preferably selected from the group of such compounds which are capable of undergoing intracellular metabolism. One preferred active ingredient for this group is the compound clodronate(13) having the following formula (II): 
Clodronate, was previously described for use in the treatment of hypercalcemia resulting from malignancy in the treatment of tumor associated osteolysis(13), and as an inhibitor of macrophages(14, 18).
Other preferred active ingredients of this group are Etidronate and Tiludronate having the following formulae (III) and (UV) respectively: 
Additional BPs having activities similar to that of clodronate are also preferred in accordance with the invention. Such BPs may be selected on the basis of their ability to mimic biological activity of clodronate. This includes, for example: in vitro activity in inhibiting phagocytic activity of phagocytic cells, e.g., macrophages and fibroblasts; inhibition of secretion of IL-1 and/or IL-6 and/or TNF-xcex1 from macrophages; in vivo activity, e.g., the ability of the tested BP to prevent or reduce restenosis in an experimental animal model such as, for example, the rat or rabbit carotid catheter injury model described in Example 1 below, or porcine model of restenosis; etc.
Another preferred group of active ingredients in accordance with the invention are the amino-BPs and any other nitrogen-containing BPs having the following general formula (V): 
wherein x represents C1-C10 alkylamino or C3-C8 cycloalkylamino, where the amino may be primary, secondary or tertiary; or x represents NHY where Y is hydrogen, C3-C8 cycloalkyl, aryl or heteroaryl The BPs belonging to this group are believed not to be metabolized and have been shown at relatively low concentrations to induce secretion of the interleukin IL-1 and cause, at relatively high concentrations, apoptosis in macrophages(18). Preferred BPs belonging to this group are for example, Pamidronate and Alendronate having the following formulae (VI) and (VII), respectively. 
Although the geminal BPs are preferred BPs in accordance with the invention, non-geminal BPs, monophosphonates of BPs termed generally as phosphonates may also be used as active ingredients in accordance with the invention.
Another preferred active ingredient is pyrophosphate having the formula (VIII): 
Pyrophosphate is preferably formulated and administered in a liposome or a polymeric particle preparation.
The composition of the invention may comprise said active ingredient either in their free acid form, complexed with metal cations such as calcium, magnesium or organic bases or may be in the form of salts or esters or they may be polymerized to yield polymers of up to 40 monomers. The salts may be sodium, potassium, ammonium or calcium salts or salts formed with any other suitable basic cation (e.g. organic amino compounds). The salts or polymers may be in a micronized particulate form having a diameter within the range of about 0.01-10 xcexcm, preferably within a range of about 0.1-5 xcexcm. The active ingredients in the compositions of the invention being either in their free acid form or salt form may be with or without water of crystallization (hydrous and anhydrous).
By a preferred embodiment, the active ingredient is encapsulated within a liposome prepared by any of the methods known in the art. Suitable liposomes in accordance with the invention are preferably non toxic liposomes such as, for example, those prepared from phosphatidylcholine phosphatidylglycerol, and cholesterol, e.g., as described below. In many cases, use of the active ingredient encapsulated in a liposome results in enhanced uptake of the active ingredient by cells via endocytosis(14, 18) (such uptake may play a role in the therapeutic effect). The diameter of liposomes used in the examples below was between 0.15 to 300 nm. However, this is non-limiting, but merely an example, and liposomes of other size ranges may also be used.
In addition, the active ingredient may be encapsulated or embedded in inert polymeric particles such as, for example, any of the microcapsules, nanocapsules, nanoparticles, nanospheres, microspheres, microparticles, etc. known in the art(19). The release of the active ingredient from such particles may be a controlled release which results, in some cases, in prolonged and enhanced effect and uptake of said active ingredient.
The pharmaceutical carrier or diluent used in the composition of the invention may be any one of the conventional solid or liquid carriers known in the art. A solid carrier for example may be lactose, sucrose, talc, gelatin, agar, etc. Where the carrier is liquid carrier it may be for example peanut oil, phospholipids, water etc. Where the solid carrier is used for oral administration, the composition may be in the form of a tablet, in the form of a hard capsule (e.g. a gelatin capsule), in the form of powder or in pellet form. Where a liquid carrier is used, the preparation may be in the form of a syrup, emulsion, liposomes, soft gelatin capsule or sterile injectable liquid such as a liposome preparation, an aqueous or non-aqueous liquid suspension or a solution(17).
The composition of the invention used for injection may be selected from emulsions, solutions, suspensions, colloidal solutions containing suitable additives, etc.
The compositions of the invention may be administered by any route which effectively transports the active compound to the appropriate or desirable site of action. By a preferred embodiment of the invention the modes of administration are intravenous (i.v.) and intra-arterial (i.a.) (particularly suitable for on-line administration). Other modes of administration include intramuscular (i.m.) or subcutaneous (s.c.). Such administration may be bolus injections or infusions. The compositions may also be administered locally to the diseased site of the artery, for example, by means of a suitable oozing/sweating balloon known in the art. Another mode of administration may be by perivascular delivery, coating of the delivery system on a balloon or a stent or by any of the other methods of cardiovascular drug delivery systems known in the art. Combinations of any of the above routes of administration may also be used in accordance with the invention.
The dosage of the active ingredient to be used also depends on the specific activity of the used active ingredient, on the mode of administration (e.g., systemic administration or local delivery), the form of the active ingredient (e.g., whether it is in the form of a drug, in a form of a polymer, whether it is encapsulated in a particle such as a liposome, etc.), and other factors as known per se.
In accordance with a preferred embodiment of the invention, treatment of an individual with the active ingredient may be for the purpose of preventing restenosis before its occurrence. For prevention, the active ingredient may be administered to the individual before angioplasty procedure, during the procedure or after the procedure as well as combination of before, during and after procedural administration.
In accordance with another embodiment of the invention, the active ingredient is administered to an individual suffering from restenosis for the purpose of reducing or treating restenosis. In such a case, the active ingredient may also be administered to the individual at different periods of time after restenosis is discovered, either alone or in combination with other kinds of treatments.
In addition, the active ingredient may be administered before any other conditions which may yield accelerated arteriosclerosis, as well as acutely after the process has begun to inhibit further development of the condition.